The objective of this proposal is to obtain a description at the molecular level of the submembrane cytoskeleton which is involved in the control of the distribution of cell surface components. We present a working model of this structure which will be tested by experiments described. Studies will be conducted on isolated protein components, membranes and intact cells. Since alpha-actinin and actin binding protein (ABP) are the key components in our model, these will be isolated for biochemical and biophysical studies and for antibody preparation. To determine how alpha-actinin binds to the membrane, we will use immunological localization and perturbation techniques with anti-actinin, binding measurements and covalent crosslinking. Binding to fractionated membrane components reconstituted into liposomes will also be tested to determine if a specific actinin-integral membrane protein linkage can be identified. The role of ABP in cell surface events will be tested in two ways. 1) Cytoskeletal ABP (insoluble) will be quantitated by a Triton extraction procedure in cells that have been treated with various morphology-perturbing agents. The results will be correlated with scanning and transmission electron microscopy, immune localization of the protein and changes in behavior of cell surface components. Cell surface behavior will be assessed not only by standard patching and capping assays, but also by measuring cooperativity of the Concanavalin A inhibition of cell surface 5'-nucleotidase, a parameter which we have shown to be sensitive to cytoskeletal perturbants. 2) The role of ABP in gelation of cytoplasmic extracts will be studied as a model for behavior of submembrane elements using proteolysis, chemical modification and fractionation procedures. Studies will be performed on sarcoma 180 cells and on sublines of the 13762 rat mammary ascites adenocarcinoma. We have a series of the latter which show varying degrees of cell surface receptor mobilities. These also have different morphological features and cell surface sialoglycoprotein contents.